The complex interplay between positive-strand RNA viruses and eukaryotic host cells is understood poorly. Although considerable information is available about the expression and function of proteins encoded by several model positive-strand RNA viruses, significant gaps exist in our understanding of the cellular structures and factors involved in virus replication, assembly and transport. In addition, relatively little is known about the critical virus-host interactions that govern the alteration or sequestration of cellular functions (such as transcription, translation, protein transport) and internal cellular structures (such as membranes) by positive-strand RNA viruses. In large part, this knowledge deficit is due to a lack of model virus-host systems in which rigorous host genetics can be applied. Thus, invaluable mutants with altered susceptibilities or responses to viruses have been isolated only rarely. This proposal centers on exploitation of the tobacco etch virus (TEV)/Arabidopsis thaliana model system to gain insight into intracellular host factors and functions involved in infection by positive-strand RNA viruses. The genome of TEV has proven to be highly amenable to modification by insertion of numerous foreign genes, including those encoding reporter proteins as well as positive and negative selectable markers. Arabidopsis offers an unbounding resource for the identification, cloning and analysis of host genes through versatile genetic approaches. This system will be used to pursue three Specific Aims. In Specific Aim l, the Arabidopsis gene RTM1 (restricted TEV movement), which limits TEV infection to inoculated organs and which was mapped recently to the top of chromosome l, will be isolated through map-based cloning methods and characterized in detail. Specific Aim 2 focuses on application of novel genetic screens to isolate and analyze additional mutants with defects in supporting TEV genome replication or movement to adjacent cells and tissues. In Specific Aim 3, the role of the cytoskeleton in TEV genome replication and movement will be explored through analysis of eight Arabidopsis mutants with temperature-sensitive defects in microtubule organization.